1. Field of the Invention
The invention is directed toward the treatment of cancer in mammals and, more particularly, to the enhancement of the clinical efficacy of traditional radiation, chemotherapy, and other techniques by the administration of specific inhibitors of the MAP kinase cascade.
2. Background of the Invention
Radiation therapy is, in many cases, the therapy of choice for the treatment of cancers, including esophogeal, mammary, head and neck, brain, prostate and certain leukemias. However, it is well-known that incomplete killing of neoplastic cells can result in the recurrance of cancer even after rigourous radiation treatment regimens are completed. Indeed, there are suggestions that some cell populations are stimulated to proliferate as a result of exposure to radiation, thus completely defeating the purpose of the treatment. Clearly, the need for more efficient methods to kill neoplastic cells persists, and a method to eliminate the occurance of cellular proliferation in response to radiation therapy would be highly beneficial.
In addition, severe side effects are often associated with radiation therapy, including fibrosis, mucocitis, leukopenia and nausea. The development of radiation therapy methods which utilize fewer exposures to radiation, or lower doses per exposure, or both, and yet which still achieve the same or enhanced levels of anti-neoplastic activity, would be highly advantageous.
Chemotherapy is also a mainstay of cancer treatment and is routinely used with success against many types of cancer. Nevertheless, certain types of cancer are not amenable to chemotherapy protocols which are currently in use. Some types of tumors simply do not respond to standard methods of chemotherapy, or respond for a time and later become insensitive, resulting in a recurrance of the cancer. New methods that enhance current chemotherapy protocols are highly desirable.
The molecular mechanism(s) by which tumor cells are killed, survive or are stimulated to proliferate after exposure to ionizing radiation are not fully understood. Several reports have demonstrated that radiation activates multiple signaling pathways within cells in vitro which can lead to either increased cell death or increased proliferation depending upon the dose and culture conditions. [Verheij et al. (1996) Nature, 380, 75-79; Rosette and Karin (1996) Science 274, 1194-1197; Chmura et al. (1997) Cancer Res. 57, 1270-1275; Santana et al. (1996) Cell 86, 189-199; Kyriakis and Avruch (1996) Bioessays 18, 567-577; Xia et al. (1995) Science 270, 1326-1331; Kasid et al. (1996) Nature 382, 813-816]. It has been shown that radiation-mediated activation of acidic sphingomyelinase generates ceramide and subsequently activates the Stress Activated Protein (SAP) kinase pathway (sometimes referred to in the literature as the c-Jun NH.sub.2 -terminal kinase (JNK) pathway). This pathway has been proposed to play a major role in the initiation of apoptosis (cell death) by radiation (Verheij et al.; Rosette et al.; Chmura et al.; Santana et al.; Kyriakis and Avruch; Xia et al.).
Likewise, the molecular mechanisms of the action of chemotherapy agents are not well-understood, particularly those processes involving specific signaling systems that impinge upon cell survival. For example, numerous signaling responses are associated with the action of the chemotherapeutic agent 1-[.beta.-D-arabinofuranosyl] cytosine hydrochloride (ara-C). These include: formation of the lipid messengers diglyceride [Kucera and Capizzi (1992) Cancer Res. 52, 3886-3891; Strum et al. (1994) J. Biol. Chem. 269, 5493-5497] and ceramide [Strum et al. (1994)]; and activation of protein kinase C (PKC) [Kharbanda et al. (1991) Biochemistry 30, 7747-7752; Riva et al. (1995) Anticancer Res. 15, 1297-1302], the p42/44 mitogen activated protein (p42/44 MAP) kinase cascade [Kharbanda et al. (1994) Mol. Pharmacol. 46, 67-72] and the SAP kinase [Saleen et al. (1995) Cell Growth Diff. 6, 1651-1658] pathway.
Experimentation has been directed toward elucidating the means by which cells survive (or even proliferate) in response to radiation exposure and chemotherapy. For example, it has been demonstrated the the antileukemic influence of ara-C is substantially augmented by pharmacological reductions in Protein Kinase C (PKC) activity [Grant et al. (1994) Oncology Res. 6, 87-99].
There are two potential protective pathways downstream of PKC: the P13 kinase pathway and the p42/44 MAP kinase pathway (Pathway 1 below). The interplay (if any) of the two pathways and their roles with respect to cellular responses to lethal agents such as radiation and chemotherapy are the subject of intense investigation and debate. Most studies have focussed on the role of the P13 pathway and these studies have shown that inhibition of P13 kinase causes apoptosis in many cell types.
Activation of the p42/44 MAP kinase cascade has been suggested to be cytoprotective versus both UV/ionizing radiation and drug treatments [Xia et al., (1995); Jarvis et al. (1997) FEBS Lett. 112, 9-14; Jarvis et al.(1997) Mol. Pharm.52, 935-947; Canman et al. (1995) Genes Dev. 9, 600-611]. Several studies have also sugested that the MAP and SAP kinase pathways may be coordinately regulated: the degree to which each is activated by a stimulus may determine the cellular fate toward differentiation, proliferation or death [Rosette et al. (1996); Kyriakis and Avruch (1996); Xia et al. (1995); Spector et al. (1997); Jarvis et al. (1997b); Cuenda et al. (1995) FEBS Lett. 364, 229-233].
With respect to the cellular response to ionizing radiation, another cellular target has been proposed to be involved. The epidermal growth factor (EGF) receptor has been shown to be activated in a dose dependent fashion in response to radiation [Schmidt-Ullrich et al. (1996) Radiation Research, 145, 81-85; Schmidt-Ullrich et al. (1997) Oncogene 15, 1191-1197]. Activation of the EGF receptor in turn activates the p42/44 MAP kinase cascade (Schmidt-Ullrich et al. 1997).
Other signal transduction pathways exist which are known to be downstream effectors of both sphingomyelinase ceramide and EGF receptor signaling. The p38-RK cascade has been described in mammalian cells as a pathway activated in response to hyper-osmotic stress, and was recently shown to be activated in response to ceramide treatment of U937 monoblasts [Jarvis et al, 1997b; Cuenda et al.(1995) FEBS Lett.364, 229-233] The p70.sup.S6 kinase and glycogen synthase kinase 3 (GSK3) are downstream effectors of PK-B/c-Akt and P13 kinase [Alessi et al (1996) J. Biol. Chem. 270, 27489-27494; Cross et al. (1995) Nature 378, 785-789] P13 kinase itself is a downstream effector of the EGF receptor, and was recently suggested to be activated in response to ultraviolet irradiation of cells [Kulik et al. (1997) Mol. Cell. Biol. 17, 1595-1606]. However, the ability of ionizing radiation to modulate the activities of these protein kinases had not been demonstrated until Carter et al. [(1998) Oncogene, 16, 2787-2796].
Activation of the p42/44 MAP kinase cascade in response to growth factors has been shown to be involved in both differentiation and proliferative responses of cells depending upon both the cell type examined and the amount of p42/44 MAP kinase activation [Kolch et al. (1991) Nature 349, 426-428; Dent et al. (1992) Science 275, 1404-1407; Pumiglia and Decker (1997) Proc. Natl. Acad. Sci.94, 448-452; Wixler et al. (1996) FEBS Lett. 385, 131-137; Traverse et al. (1994) Curr. Biol. 4, 694-701; Whalen et al. (1997) Mol. Cell. Biol. 17, 1947-1958]. Acute activation of the MAP kinase cascade by growth factors has been shown to potentiate proliferation, whereas chronic elevation of MAP kinase activity has been demonstrated to be cytoprotective against irradiation (Canman et al. 1995) and to inhibit DNA synthesis potentially via induction of the cyclin dependent kinase (cdk) inhibitor protein p21.sup.Cip-1. [Pumiglia and Decker (1997); Wixler et al. (1996); Traverse et al. (1994); Whalen et al. (1997); Lloyd et al. (1997) Genes and Dev. 11, 663-677; Fan et al. (1995) J. Cell. Biol. 131, 235-242; Missero et al. (1996) Genes and Dev. 10, 3065-3075; Macloed et al. (1995) Genes and Dev. 9, 935-944; Liu et al (1996) Cancer Res. 56, 31-35; Deng et al. (1995) Cell 82, 675-684] The cdk inhibitor protein p21.sup.Cip-1 can also be induced in response to radiation exposure of cells, and cultured fibroblasts from p21.sup.Cip-1 `knock-out` mice which cannot express p21.sup.Cip-1 have been shown to be more radiosensitive than wild type cells, demonstrating that expression of this molecule is cytoprotective against radiation [Missero et al. (1996); Macloed et al. (1995); Liu et al (1996); Deng et al. (1995)] The mechanism by which p21.sup.Cip-1 is induced in response to irradiation of cells has been proposed to be via the function of p53 which is able 10 to sense DNA damage, and not by p42/44 MAP kinase signaling. This data would appear to preclude signaling via the p42/44 MAP kinase cascade as a player in the radiation-mediated induction of p21.sup.Cip-1 [Missero et al. (1996); Macloed et al. (1995); Liu et al (1996); Deng et al. (1995); Van den Heuvel and Harlow (1993) Science 262, 2050-2054; Lees et al (1993) Oncogene 8, 1593-1602; Brugarolas eta 1. (1995) Nature 377, 552-557; Freemerman et al. (1997) Leukemia 11, 504-513; Akiyarna et al. (1997) Cancer Res. 57, 57, 1495-1501; Mothersill et al (1995) Radiation Res. 142-181-187; Bernhard et al (1995) Radiation Environ. Biophys. 34, 79-83; Muschel et al (1997) Vitam. Horm. 53, 1-25; Johnson et al. (1994) Mol. Carcinogenesis 11, 59-64; Michieli et al. (1994) Cancer Res. 54, 3391-3395]. However, other reports [Carter et al. (1998)] have demonstrated radiation induction of p21.sup.Cip-1 in a MAP kinase dependent fashion in cells which express non-functional p53.